Examination of objects of macromolecular size

ABSTRACT

In a method and apparatus for examining samples comprising individual objects (28) of macromolecular or similar size, or smaller, an instrument element (14) comprises a substrate (16) overlaid with a thin film layer (18) of a material that is electrically conductive and/or at last partly optically opaque. A discontinuity comprising an aperture (26) or an asperity is formed in or on the element (14) in a known location, and the sample (28) is brought to this discontinuity so that it is not necessary to search for the sample by scanning. Energy (e.g. electrical energy or light) is applied to the element (14) and, with the latter and the sample in intimate association, e.g. with the sample inside the aperture (26), changes in the radiation from the sample site resulting from the presence of the sample are detected.

This invention relates to methods and apparatus for examining individualobjects the size of which is of the general order of magnitude ofmacromolecules and their aggregates, or smaller. These objects mayconsist of particles, or of other materials or structures of generallysimilar dimensions. Where they are particles, they may in fact actuallybe macromolecules, for example enzymes or other proteins, or biologicalmacromolecules or other larger structures such as viruses. The fieldcovered by the invention does not however exclude the examination ofparticles of generally sub-micron size. Objects for examination, orbeing examined, will sometimes herein be called "samples", forconvenience.

Current techniques, such as scanning tunnelling microscopy, atomic forcemicroscopy and scanning near field optical microscopy, are already wellknown, and have considerably lowered the limit of molecular resolutionthat is now possible. In all these techniques, the image is built upfrom a scanned signal which is generated by interaction of the structureof the sample particle with a probe tip having dimensions comparablewith that of the sample.

In scanning tunnelling microscopy (STM) an ultra-fine, chemically etchedelectrode is brought very close to the sample, so that electrons canpass by quantum tunnelling across the free space between the electrodetip, or part thereof, and the surface of the sample. The samples aremounted on a conductive substrate, and the probe is scanned across thesubstrate in order to find a sample for examination. Piezoelectrictransducers are used to control the movement of the probe. Onedisadvantage of STM is that the sample must be sufficiently electricallyconductive, and with many types of particle, particularly biologicalparticles, this requires pretreatment of the sample macromolecule inorder to improve its conductivity. This has been achieved by using metalshadowing or doping with conductive salt ions; such treatment canhowever alter the characteristics of the sample, so that in some casesit becomes self-defeating.

Another, somewhat similar, device is the scanning capacitancemicroscope, which measures the capacitive, changes associated withsample structures but on a much lower resolution. It can operate to thesame order of magnitude of sample size as conventional opticalmicroscopy, or larger.

In atomic force microscopy (AFM), an ultra-fine, very lightly springloaded probe tip is scanned across an area containing a sample, and thenacross the sample itself. The movement of the tip as it is deflected byrepulsion between the Van der Waals forces between the tip and thesample is monitored so as to generate a topographical image of thesurface of the sample. A major disadvantage of AFM is that with mostbiological macromolecular structures, particularly in solution, theforces generated by the probe tip will tend to damage or destroy thesample. The AFM technique is therefore somewhat limited in scope, and,as is also generally true of STM, it is really only suitable for usewhere the sample can be shadowed, i.e. coated, with metal.

Electromagnetic radiation can be greatly amplified when interacting withstrongly curved parts of a surface due to quasi-static concentration offield lines (the so-called "lightning rod" effect). This effect isresponsible for the development of surface-enhanced Raman scattering orroughness-induced electrical breakdown. It is also responsible for thestrong elastic light scattering associated with microscopic holes in afilm (apertures) or protrusions (asperities) from a surface, a processclosely related to Mie scattering from small spheres which act as "shortoptical antennae" (SOA). The degree of scattering from these SOA isrelated to their size (curvature) and dielectric properties.

The method employed in a scanning near field optical microscope (SNOM)is a variant on the STM technique, and is essentially optical incharacter. The SNOM includes a component which serves as an opticalsource of suitably small dimensions and which performs an activefunction in the examination process. Such a component will be found inwhat will be generally called an "instrument element" in thisdescription. In the SNOM, the instrument element can comprise asubstrate of a suitable waveguide material, having a very thin layer ofgold deposited on one surface. The gold is so applied that it has smallapertures or asperities (generally no larger than 100 nm), which act ascentres for light scattering. In other words, the apertures orasperities act as short optical antennae. The effect of this scatteringis that some radiation leaks from the apertures or asperities in thegold film, the intensity of the scattered radiation being dependent onthe size of the aperture and on the dielectric properties of thematerials. The scattered light emanating from the apertures orasperities is easy to detect using conventional optics. The SNOM carriesout optical topographical imaging of samples by monitoring the changesin intensity of the light scattered from the aperture or asperity as thesample is brought into very close proximity to the radiating near fieldemanating from the aperture or asperity. In the near field region, theelectric field of the radiation is severely damped by the approachingsample, and this sensitivity is used for the topographical imaging ofthe sample.

A major drawback in all of these known methods is that before a samplecan be examined, it must first be found. Thus, a region of the samplepreparation which contains an object of interest must first beidentified, and this can be difficult. Although attempts have been made,in connection with both STM and AFM, to associate these types ofmicroscope with electron microscopes or conventional opticalmicroscopes, for example, in order to take advantage of the wider rangeof scan of these more conventional instruments, searching for a suitablespecimen can still waste a very large amount of time, because STM, AFMand SNOM inherently have a small width of scan. SNOM also requires themounted sample to be positively approached towards an aperture orasperity in the gold film. As also with STM and AFM techniques, themounting or immobilising of the sample in SNOM can result in damage tothe sample. At the same time it is not possible to bring the opticalprobe close enough to the sample to be at an optimum distance from itwithout risking untoward close contact,or even impact, between thesample and the probe. Even so, the distance between the sample and theaperture has to be very closely controlled, and as with the othertechniques, the equipment involved is somewhat complex and expensive.

Another disadvantage found with the current techniques is that, whereasresolution normal to the plane of the aperture in the instrument elementdefining the site at which examination of the sample is to take place(vertical resolution) is high, lateral resolution is much lower. In thecase of STM and AFM instruments, it is not possible to produce a probetip narrow enough to produce lateral resolution comparable to the highdegree of vertical resolution which is possible. This drawback is moremarked in the case of the SNOM, and the smallest probe diameter is ofthe order of 30 nm. A further disadvantage is that interaction betweenthe sample particles and the radiation scattered at the aperture may ofnecessity be relatively poor.

Another method of detecting individual cells (1-5 μm and larger) is thatof conventional flow cytometry, in which cells are hydrodynamicallycaused to pass through an optical scattering volume (laser beam) foranalysis. As with other conventional optical systems, the resolutionavailable with flow cytometry is limited by diffraction effects, whichlimit the degree of beam focussing that can be obtained.

According to one aspect of the invention, a method of examining samplingcomprising individual objects of macromolecular size or smaller being anon-scanning, non-image-forming method, from which measurement ofdistances is absent and which comprises the steps of:

(i) bringing a sample into proximity with an instrument elementcomprising at least one thin film layer having a discontinuity in aknown or identifiable location;

(ii) exciting the instrument element with electromagnetic radiation towhich the material of said layer is substantially opaque, so as to causea detectable signal to emanate from the discontinuity;

(iii) causing the sample to move into intimate association with thediscontinuity so that the sample and the discontinuity then interactwith each other to produce changes in said detectable signal; and

(iv) continuing to excite the instrument element with said radiationwhile detecting said changes.

Where the discontinuity is an aperture formed through the film layer,the "intimate association" referred to above consists in the presence ofthe sample, or part of the sample, in the aperture itself. Accordingly,samples suitable for examination with this arrangement will generallyconsist of particles and other bodies which can be brought into theaperture. The aperture is preferably larger than at least one expecteddimension of the sample, but of a similar order of magnitude. However,the arrangement can also be used for study of the interaction betweentwo samples, one of which may for example be partly in the aperture andthe other one close to it.

If the discontinuity is an asperity projecting from the film layer, thesample may or may not be of such a configuration that it could bemounted in or pass through an aperture. Use of asperities is especiallysuitable where the sample consists of a membrane or analogous structure.The "intimate association" can take any suitable form, depending onrequirements: the sample may for example be in actual contact with theasperity, or almost in contact with it. In one type of practicalembodiment, the portion of the film layer bearing the asperity can becoated with a sample membrane, with other samples, of any kind, thenbeing brought into intimate contact with the latter at the asperity sothat these samples can be analysed in terms of their interaction withthe membrane by detection of the changes in radiation emanating from theasperity in the presence of such a sample.

In general, the instrument element with its discontinuity is preferably,though not necessarily, arranged in a fixed position, so that therelative movement between sample and discontinuity preferably consistsin conveying or attracting the sample towards the discontinuity and intothe appropriate intimate association with it, using electrophoresis orany other suitable means.

Preferably, the method also includes the further step of applying anenergy field or fields to the discontinuity in such a way as to modulateor modify the behaviour and/or the structure of the sample. Thisadditional or modulating energy may or may not be of the same kind asthe basic energy which is applied to cause the detectable signal tooccur at the discontinuity in the first place. This basic applied energymay be electrical energy or electromagnetic energy such as light. In thelatter case, the aperture or asperity acts as a short optical antenna.

The invention, in a second aspect, is directed to apparatus forperforming the method of the invention, said apparatus comprising aninstrument element that comprises at least one thin film layer, having adiscontinuity in a known or identifiable location; means for causingrelative movement between a sample and the discontinuity and forbringing them into intimate association with each other; means forexciting the instrument element with electromagnetic radiation to whichthe material of said layer is substantially opaque, so as to cause adetectable signal to occur at the discontinuity; and detecting means fordetecting said signal and changes therein, without scanning or imageforming, the apparatus defining means connecting the discontinuity withthe detecting means whereby the latter can receive said signal.

Considering the case where the discontinuity is an aperture, it is animportant feature of the invention that the sample is actually broughtinto close proximity to, or preferably actually into the interior of theaperture itself. Thus, instead of examination of a sample being possibleonly when it lies in the near field region outside an aperture, intowhich radiation is scattered from the latter, as in conventional SNOMpractice, the sample can actually be brought into the intimateassociation, discussed above, with the aperture as well as the nearfield. The invention thus enables advantage to be taken of tunnellingeffects in addition to the near field optical effects which characteriseknown methods based on scanning. Thus the detectable signal may takemore than one form, e.g. changes in electromagnetic radiation orelectrical parameters at the discontinuity (collectively referred tobelow as "radiation" for convenience).

The net result is that the sensitivity of the apparatus is greatlyincreased, since the radiation field within the aperture, of a similarorder of physical size to the particle, will have a profound effect onthe behaviour of the radiation, and therefore a substantially increasedeffect on the radiation which "leaks" from the aperture. These changescan be quite readily detected by conventional means. Similarconsiderations apply where the discontinuity is an asperity.

In alternative embodiments, in which light is used as the source ofenergy, the film layer is then made at least partly opaque to light, thesubstrate being able, either by being porous or otherwise, to transmitlight, so that light applied through the substrate leaks from it throughthe aperture in the film layer. In this case, as indicated above, thepresence of the particle within the aperture itself will cause aconsiderably more marked disturbance of the light leaking from theaperture. Indeed, in this case conventional optical equipment will inmost cases be sufficient to detect the changes in the visible lightcaused by the presence of the particle in the aperture.

The invention is particularly well adapted for the examination ofsamples in solution. An electrical potential is applied across theinstrument aperture so that the solution, containing the sample in freesolution in a solvent, migrates towards it, for example byelectrophoresis, and particles or other samples in solution can thusthemselves migrate to the location of the aperture and so into thelatter. For this purpose, the substrate is porous and its electricalconductivity is low enough, i.e. it is a sufficiently good insulator,for the aperture and porous channel to act as a preferred leakage path.

The invention eliminates the need to scan a surface over which sampleobjects are attached, firstly in order to find a sample for examinationin the first place, and secondly to examine the sample once it has beenfound. Instead, the sample is deliberately directed into a singlepredetermined location, where it is then examined. It follows that aplural number of samples can be simultaneously examined, using theirseparate interactions with a corresponding number of apertures and/orasperities.

Where this predetermined location consists of an aperture in or throughthe instrument element itself, the sample is examined by observing theinteraction between the applied energy and the sample inside theaperture (or that part of it that is inside the aperture as the particleelectrophoreses through the latter). The aperture is preferably of knowndimensions, so that the detection volume (i.e. the volume of theaperture) is highly specific and suitable for the size and associatedfield of a sample passing through it. This results in a substantialreduction in unwanted "noise", and hence a very significant improvementin signal-to-noise ratio, resulting in turn in a substantial improvementin resolution and accuracy of the usable output signals. Theseimprovements are also evident where the discontinuity is an asperity.The invention also offers other advantages, some of which have alreadybeen mentioned above.

The invention is intended primarily for use on samples in which theinteraction between the sample and the applied energy, e.g. light, isgoverned by quantum mechanics. It should however be understood that theinvention is also applicable to the examination of microscopic objectslarge enough for more conventional laws to be applied. Many viruses forexample, larger than about 30 nm in size, fall into this category.

Analysis of the object (i.e. particle, molecule or structure, asdiscussed earlier in this specification) in the aperture can be carriedout using time-resolved optical techniques (fluorescence), or any otheranalytical method operating in the time domain, and in which the objectis specifically modulated, optically or electrically, so as to beoptically or electrically phase locked into any given detectionprinciple.

A few examples of the application of the invention will now bedescribed, by way of example only, with reference to the accompanyingdrawings, all of which are highly diagrammatic and not to scale. In thedrawings:

FIG. 1 shows in cross section part of the instrument element of one formof apparatus according to the invention, in which particles are causedto migrate through the instrument element at a known location, and areexamined by observing the interaction between them and applied light;

FIG. 2 is a cross section through part of the instrument element inwhich the particle is held stationary in the aperture;

FIG. 3 illustrates one use of the invention where the discontinuity isan asperity acting as a short optical antenna;

FIG. 4 illustrates the use of piezoelectric effects to produce anapplied energy field;

FIG. 5 is a cross section of part of an instrument element incorporatinga convergent waveguide;

FIG. 6 illustrates a use of the apparatus for studies at a liquid-liquidinterface; and

FIG. 7 illustrates a use of the apparatus in DNA sequencing analysis.

The applied energy used in the method and apparatus of the invention isany form of electromagnetic radiation, and in FIGS. 1 to 3 thiselectromagnetic energy is light. In FIG. 1, an instrument element 60separates from each other two receptacles 10 and 12. The receptacle 10contains a solution, typically an aqueous solution (though any suitablesolvent, liquid or gaseous, can be used), in which particles 62 to beexamined are in free solution, in the solvent. The element 60 comprisesa porous, translucent, electrically insulating substrate membrane 66, onthe front face of which an opaque film layer 66 is deposited by anysuitable means. The layer 66, which may typically be of metallic gold,may have any suitable thickness, typically of the order of 20 nm. Atleast one hole 68 is formed, by any known method, through the film layer66, at a known location in the latter. The hole 68 and the porosity ofthe membrane 64 immediately behind it together define what is referredto herein as an "aperture", or path, through the element 60. Thisaperture is indicated at 70 in FIG. 1.

If desired, the instrument element may have a number of such apertures,all in known locations but of different sizes so that the apparatus caneffectively handle a variety of different kinds of particles ofdifferent sizes.

A voltage is applied between the two receptacles 10 and 12, such as tocause the solution to migrate electrophoretically from the formertowards the later through the hole 68.

It will of course be understood that close interaction between theparticle and the aperture may be obtained by any suitable techniqueusing a selective physical or chemical motive force. Examples includeosmosis, diffusion and centrifugation. The preceding examples, thesolution is thus caused to migrate electrophoretically in the receptacle10 to the instrument element 60, so that the particles 62 pass throughthe aperture in the latter.

The translucent membrane 64, which is preferably transparent, is edgeilluminated, for example by means of a laser 71, at an angle ofincidence such that it acts as a waveguide, as indicated by thedirectional lines 72 which represent light paths within themembrane-substrate layer. Although the light following the paths 72 isgenerally constrained within the membrane 66, some of it will leakoutwardly through the hole 68 to give near-field light leakage orscattering as indicated at 74. The light paths 72 are at grazingincidences only, so that light does not penetrate the film layer 66,except at the holes 68 which act as the scattering centres.

As a particle 62 approaches the aperture or porous path 70 underelectromotive force, it will start to interact with the electromagneticfield associated with the aperture which acts as a short optical antenna(SOA) according to the well-known principles explained above. The effectof this is that the scattered light 74 will change in respect of atleast one optical parameter, such as intensity, polarisation, phase,spectral content or fluorescence. This effect will increase as theparticle approaches the hole 68, and be strongest when the particle isactually in that hole. The scattered radiation 74, modified by theparticle, therefore represents an output signal which can be received byany suitable optical receptor. In this example, the latter is aconventional microscope objective lens 76, typically of ×40magnification.

The optical signal from the objective lens 76 may then be processed andanalysed by any suitable optical and/or electro-optical instrumentation,not shown.

It should be noted that this process again involves interaction betweenthe light itself, i.e. the electromagnetic field associated with theaperture 68, acting as an SOA, and the electrostatic or dielectriccharacteristics of the particle. Accordingly, the porosity of themembrane 64, and, once again, the size of the hole 68, are chosen totake into account the electrostatic double layer surrounding theparticle and forming, in electrostatic terms, part of the particleitself. The hole 68 is typically from 20 to 200 nm in diameter. We shallreturn later herein briefly to the significance of this double layer.

The opaque layer 66 may be made unsupported, being back-illuminated atsuitable grazing incidence angles such that light does not pass directlythrough the hole 68 but only escapes from it by virtue of smallaperture/asperity scattering effects. However, where the layer 66 issupported by a substrate 64, the latter may be of any suitable materialwhich is porous to the macromolecule, for example hydrophilic orhydrophobic gel, compatible with the solvent in which the particles 62are carried in free solution. Another possible material for the membraneis controlled-pore glass. The objective 76 is focussed on one particularselected hole 68, and the film layer 66 may be formed with no otherperforations. It may however have a large number of perforations, andFIG. 1 shows more than one. Particles passing through any of these otherperforations are of no account. Alternatively, of course, perforationsof different sizes, each in a known location onto which the objective 76can be focussed beforehand, may be provided for the examination ofparticles of different sizes.

Reference is now made to FIG. 2, in which the instrument element, 90,does not define a path for a particle through the element itself. Herethe aperture consists merely of the hole 68 formed in a film layer 66,generally similar to that described with reference to FIG. 1 and mountedon a transparent waveguiding substrate layer 92, which need not beporous but may merely consist of optical glass. The substrate layer 92is again edge illuminated at grazing incidence, so that the only lightpassing out through the hole is the leakage radiation 74. The glasssubstrate 90 is suitably treated by any conventional method foractivating it, for example (where it is of glass) by an immobilisationtechnique such as silanisation such as to enable the samples, forexample biological macromolecules, to be simply attached to the glass.Thus, with the substrate 92 of glass and the opaque layer 66 of gold, ifthe resulting element 90 is treated by a liquid or vapour phasesilanisation technique, since gold reacts poorly to such immobilisationchemistries, the element 90 will be activated substantially only on theparts of the glass surface exposed within the hole or holes 68. Thesample macromolecules, 96 in FIG. 2, will therefore tend to becomeattached, by a silane immobilising ligand 98, within the holesthemselves. Thus, as before, a signal receptor such as the objective 76in FIG. 1 can be focussed on one particular hole 68, the particlesadhering anywhere else on the element 90 being of no account. Becauseparticles will preferentially attach themselves in holes 68, it ismerely necessary to focus on to one of the latter, without the need forany scanning to locate a particle for examination. In this connection,it should be noted that even if some attachment of particles to the goldsurface does occur, these will cause no interference since the usefuloutput signals are generated only by structures associated with the hole68.

When a particle is trapped in a specific location in this way,experiments can be carried out for a number of different purposes on thestationary particle at leisure. In particular, the arrangement shown inFIG. 2 may be used for the detection and analysis of other bodiesinteracting with the sample or samples located in the aperture 68. Itfollows that a sample, such as a particle, in the aperture 68 can beaugmented with another material, or composite, of suitably small size,which may interact with the sample in such a way as to cause changes totake place in its optical and/or electrical properties. These changescan then be detected and analysed.

FIG. 3 illustrates another embodiment in which the effects ofinteraction can be studied. In this case the transparent substrate, 100,again has a thin gold film, 102, on one of its surfaces, but here thediscontinuity is an asperity instead of an aperture. The asperity is aprojection 104 on the outer side of the film 102, which is coated with alayer of a sample material, such as a biological membrane 106, with thislayer covering the asperity 104. Other samples, such as a membrane 108or a particle 110, are brought by electrophoresis or otherwise intocontact with, or very close proximity to, the membrane 106 in the region112 where the latter overlies the asperity 104, such that in this regionthere is interaction between the transported sample 108 or 110 and themembrane 106.

Light is applied to the instrument element 100, 102, for example via thesubstrate 100 as already described in connection with FIGS. 1 and 2. Thefilm 102 is thin enough to be only partly opaque to this light so thatsome light passes through it, the remainder of the light being reflectedby the film. The asperity 104 acts as a short optical antenna, causingscattering of both transmitted and reflected light.

When interaction occurs between a sample 108 or 110 and the biologicalmembrane 106, the resulting changes in the light emanating from the SOAasperity 104 can be detected. This may be done, for example, using atransmitted-light lens 114 on the same side of the instrument element asthe asperity, or a reflected-light lens 116 on the other side of theelement, since both the transmitted and reflected light will be affectedby the interaction between the samples.

The configuration of the asperity 104, and its dimensions, can be chosento suit the particular application for which it is to be used.

Another example, using the instrument element 90 of FIG. 2, isillustrated in FIG. 7. It should be noted that the arrangement seen inFIG. 3 could, however, be used instead. In FIG. 7, a polymerase molecule120 is attached by a ligand 98 in the aperture 68 over the waveguidesubstrate 92. A single-stranded DNA molecule 122, with a primer sequence124 base-paired on to it in a known manner to initiate polymerisation,is brought to the polymerase 120. The progress of the resultingpolymerisation with nucleotide bases 124 and fluorescently-labelledbases 126, giving the duplex DNA structure 127, can then be observed andanalysed due to the changes in the light emanating from the aperture 68,light being supplied to the substrate 92 as before.

A chemical or biochemical passivation or modification layer 128, ofsuitable composition, may be applied over the outer surface of the metallayer 66 in order to improve the specificity of binding of the targetDNA molecule 122, and/or to reduce non-specific binding. It should benoted that such a layer 128 can be employed in all variants of themethod or apparatus of this invention where these effects may berequired.

The instrument element can be configured in a miniature waveguide form,e.g. as a slab or other waveguide, or in a fibre optic form. In thelatter case it is, for example, capable of being used as a remote,multiplexable fibre optic sensor which can be incorporated, if required,into a network.

FIG. 5 shows one example in which the substrate 130 of the instrumentelement, having a metal film layer 66 and an aperture 68 as before, isof glass so as to act as a monomode optical waveguide. A convergentsurface profile 132 is formed on the rear face of the substrate 130leading to the aperture 68. This profile acts as an efficient, tapered,local waveguide, enhancing containment of the incident light andconcentrating the latter at the aperture 68 while reducing interfaciallosses.

The convergent profile 132 terminates in a hole 134 aligned with thehole 68 in the layer 66 so that the aperture 68 itself comprises thesetwo holes, which are preferably drilled by high-energy electron beamlithography after suitable masking (a hole-forming technique which canbe used for any of the apertures in the various embodiments of theinvention in which the discontinuity serving as the working site is anaperture).

The concentration of light at the aperture, together with the reducedlosses, may produce flare effects involving sub-wavelength tunnellingeffects which can enhance the observable changes that supply theinformation required when a sample is present at the aperture.

The profile 132 can be of any desired shape, e.g. spherical or conical,and may be formed by etching.

Another possible use of apparatus according to the invention is in thestudy of a stable liquid/liquid interface 140, FIG. 6, or in the studyof the behaviour of molecular objects at such an interface. FIG. 10shows two such molecules, 142, suspended at the interface within theaperture 68 of an instrument element 143 which (in this example) happensto be similar to that shown in FIG. 5, but which need not have a taperedwaveguide section. A first liquid solvent 144 is above the element 143,and a second liquid solvent 146 below it. These solvents will be chosenat least partly so that their physical characteristics permit the stableinterface 140 to form in the aperture 68. They, and/or the samples, willalso be chosen so that the latter are ormphiphilic.

Referring now to FIG. 4, an applied energy field can be createdpiezoelectrically. In FIG. 4, the substrate 150 of the instrumentelement is of an optically-transparent piezoelectric material to which apair of electrodes is fitted, the electrodes being connected through apiezoelectric resonator circuit or driver 152. Depending on thepiezoelectric polarity, these electrodes are mounted so as to transmitimpulses through the substrate 150 either transversely (electrodes 154)or longitudinally (electrodes 156 in phantom lines). In the former casethe whole of the metal layer 66 may serve as an electrode.

With a sample 158 mounted in the aperture 68 in the layer 66, light 72is transmitted through the substrate as described with reference toFIGS. 1 and 2. In addition, the driver 152 is energised so as tosuperimpose piezoelectric energy on the substrate, at a frequency whichexcites the sample at an acoustic (audio or ultrasonic) frequency andmodulates the observed optical output. This produces the well-knownphenomenon of Brillouin scattering, which depends on the interaction ofthe applied light and acoustic energy and also on the characteristics ofthe sample which determine the optical output. Data on suchcharacteristics can in this way be obtained.

The electrostatic double layer of a macromolecular particle has beenmentioned above. This is part of its characteristic electrostatic field.In the case of certain biological macromolecules, small differences inprimary structure lead to significant differences in their function.Current theories on protein, enzyme and other macromolecule structureand function suggest that secondary and tertiary structure isresponsible for generating large electrostatic fields, which may extendto the equivalent of several diameters of the molecule itself. This istrue for example in the case of some proteins, especially enzymes.Theoretical studies of these electrostatic fields have indicated a rolein the capture of specific substrate molecules at relatively greatdistances by an enzyme macromolecule. The fields are generally toroidalin shape, and it has been shown that they have their poles centred onthe active site of the enzyme, and that they act not only to facilitateinteraction between the active site and the substrate molecule, but alsoalign the substrates on their approach to the active sites. Thestructure and shape of these fields are accordingly likely to be highlycharacteristic of a specific enzyme, and the present invention providesa useful and versatile method of studying and exploiting thesecharacteristic electrostatic fields in detail.

It should be noted that the invention is not confined to the case wherethe samples are in solution. Where they are, however, the solvent neednot be water or even liquid, but the solution may take any form known tophysical chemistry in which the sample particles can be caused tomigrate to the examination site, either electrophoretically or under anyother selective physical or chemical motive force. One example of such asolution is a gel.

Where the energy used for observation is light, i.e. in opticalembodiments, a number of different optical parameters may be studied atthe aperture or asperity, notably Brillouin scattering (alreadymentioned), intensity, fluorescence, polarisation and absorption. Theintensity of light scattered at the aperture or asperity (thediscontinuity) is modulated by the mass or dielectric of analyte samplesin the vicinity of the discontinuity if the latter acts as a scatterer.As to fluorescence, aspects of this that are available for study includeintensity of fluorescence, shifts in excitation or emission wavelengthsor profiles, decay patterns of fluorescence, and Raman scattering.Fluorescence may be observed by conventional spectroscopy using theapparatus of the invention.

Where a sample is optically active, changes in their polarisation may Dedetected, including depolarisation.

Absorption of light by a sample can manifast itself in the form ofchanges in intensity of the incident light, and can cause local heatingaround the sample (e.g. a molecule), so changing the local opticalenvironment, which may then be analysed, for example by a photothermaltechnique. Again, absorption may be studied using conventionalspectroscopy using the apparatus of the invention.

The thin film, which in the examples given above is of gold, can inpractice be of any material that is suitable in terms of electricalconductivity and/or opacity, and which may or may not be a noble metal.

Besides the embodiments of the method and apparatus described above, theinvention can be employed in a variety of other configurations and for avariety of other purposes, involving simple examination and analysis ofsamples; modification of the behaviour and structure of individualsamples with examination and analysis of such modifications and/or theireffects; and study of the interaction between a plurality of samplesand/or between one or more samples and their environment.

Whatever embodiment of the invention is employed, and for whateverpurpose, it will be seen that, since the need for scanning to find asample is avoided, and since consequently the examination site can bepredetermined in advance, it is also possible to calibrate that site inconjunction with the parts of the apparatus that receive the signals andanalyse them to give information about the sample. This will tendgreatly to decrease the possibility of inadvertent error, such as mayoccur when the location of the site is randomly determined only by thefact that a sample happens to have settled there.

It should however be noted that use of the method and apparatus of thepresent invention does not necessarily preclude its combination forcertain purposes with known forms of equipment. For example, theapparatus of FIG. 2 may be incorporated in a scanning tunnellingmicroscope, so that the STM probe can be used to pick up a molecule andplace it on the sample macromolecule already attached in the hole 68, sothat the resulting effects can be observed. The probe releases themolecule being carried to the site simply by changing the voltage to theprobe. Similarly of course the added molecule may be removed.

We claim:
 1. A method of examining samples comprising individual objectsof macromolecular size or smaller the method being a non-scanning,non-image-forming method from which measurement of distances is absentand which comprises the steps of:(i) bringing a sample into proximitywith an instrument element comprising at least one thin film layerhaving a discontinuity in a known or identifiable location; (ii)exciting the instrument element with electromagnetic energy to which thematerial of said layer is substantially opaque, so as to cause adetectable signal to occur at the discontinuity; (iii) causing thesample to move into intimate association with the discontinuity so thatthe sample and the discontinuity then interact with each other toproduce changes in the detectable signal at the discontinuity; and (iv)continuing to apply the said energy while detecting said changes,andwherein, the discontinuity consists of an asperity on the film layer,step (iii) comprises bringing the sample into contact, or almost intocontact, with the asperity.
 2. A method of examining samples comprisingindividual objects of macromolecular size or smaller the method being anon-scanning, non-image-forming method from which measurement ofdistances is absent and which comprises the steps of:(i) bringing asample into proximity with an instrument element comprising at least onethin film layer having a discontinuity in a known or identifiablelocation; (ii) exciting the instrument element with electromagneticradiation to which the material of said layer is substantially opaque,so as to cause a detectable signal to occur at the discontinuity: (iii)causing the sample to move into intimate association with thediscontinuity so that the sample and the discontinuity then interactwith each other to produce changes in the detectable signal at thediscontinuity; (iv) continuing to apply the said energy while detectingsaid changes; and, (v) applying to the sample and/or the instrumentelement, at least while step (iv) is being performed, an energy fieldsuch as to modify or modulate the behavior of particles and/orstructures within the sample.
 3. A method according to claim 2, whereinapplication of the said energy field comprises modulating the energyapplied in step (ii).
 4. A method according to claim 2, wherein the saidenergy field is applied as a different form of energy from saidelectromagnetic radiation.
 5. A method of examining samples comprisingindividual objects of macromolecular size or smaller, being anon-scanning, non-image-forming method, from which measurements ofdistances is absent and which comprises the steps of:(i) bringing asample into proximity with an instrument element comprising at least onethin film layer of a material substantially opaque optically and havinga discontinuity in a known or identifiable location; (ii) exciting theinstrument element with light so as to cause a detectable optical signalto occur at the discontinuity; (iii) causing the sample to move intointimate association with the discontinuity so that the sample and thediscontinuity then interact with each other to produce changes in thesaid signal; and (iv) continuing to :apply the said energy whiledetecting said changes,and further including the step of applying to thesample and/or the instrument element, at least while step (iv) is beingperformed, a field of acoustic or ultrasonic energy for excitation ofthe sample such as to modify or modulate the behavior of particlesand/or structures within the sample, step (iv) comprising detectingchanges in light emitted from the immediate vicinity of the sample andresulting from the said acoustic or ultrasonic excitation.
 6. Apparatusfor use in performing the method of claim 2 comprising:an instrumentelement comprising at least one thin film layer having a discontinuityin a known or identifiable location; means for causing relative movementbetween a sample and the discontinuity and for bringing them intointimate association with each other; means for exciting the instrumentelement with electromagnetic radiation to which the material of saidlayer is substantially opaque, so as to cause a detectable signal tooccur at the discontinuity; and detecting means for detecting saidsignal and changes therein without scanning or image forming, theapparatus defining means connecting the discontinuity with the detectingmeans whereby the latter can receive said signal,wherein thediscontinuity is an asperity on the film layer.
 7. Apparatus for use inperforming the method of claim 2, comprising an instrument whichincludes at least one thin film layer having a discontinuity in a knownor identifiable location; means for causing relative movement between asample and the discontinuity and for bringing them into intimateassociation with each other; means for applying light to the instrumentelement whereby to excite the instrument element, so as to cause adetectable signal to occur at the discontinuity; and optical detectingmeans in line of sight with the discontinuity for detecting said signaland changes therein, without scanning or image forming, wherein theinstrument element further includes a substrate on which said at leastone film layer is overlaid, said exciting means comprising a lightSource, the substrate being translucent for conveying light from saidsource to the discontinuity.
 8. Apparatus for use in performing themethod of claim 2, comprising:an instrument element comprising at leastone thin film layer having a discontinuity in a known or identifiablelocation; means for causing relative movement between a sample and thediscontinuity and for bringing them into intimate association with eachother; means for exciting the instrument element with electromagneticradiation to which the material of said layer is substantially opaque,so as to cause a detectable signal to occur at the discontinuity; anddetecting means for detecting said signal and changes therein withoutscanning or image forming, the apparatus defining means connecting thediscontinuity with the detecting means whereby the latter can receivesaid signal, and further including modulating means connected with theinstrument element, for applying to a sample at the discontinuity anenergy field for modulating said electromagnetic radiation whereby tomodify or modulate the behavior of particles and/or structure within thesample.
 9. Apparatus for use in performing the method of claim 2,comprising:an instrument element Comprising at least one thin film layerhaving a discontinuity in a known or identifiable location; means forcausing relative movement between a sample and the discontinuity and forbringing them into intimate association with each other: means forexciting the instrument element with electromagnetic radiation to whichthe material of said layer is substantially opaque, so as to cause adetectable signal to occur at the discontinuity; and detecting means fordetecting said signal and changes therein without scanning or imageforming, the apparatus defining means connecting the discontinuity withthe detecting means whereby the latter can receive said signal, andfurther including energy applying means connected with the instrumentelement, for imposing on the instrument element, and on a sample at thediscontinuity, a field of a different form of energy from saidelectromagnetic radiation, whereby to modify or modulate the behaviourof particles and/or structure within the sample.